Project summary Chromatin-based DNA damage response (DDR) is essential for protecting cells from genome instability, a hallmark of cancer. Histone H2A family is an important unit of chromatin, where DNA double strand breaks occur and DNA damage response is initiated. The H2A family has multiple variants, which are diverse in sequence homology. Notably, macroH2A, an H2A variant occupies a quarter of the autosomal genome, is implicated in DNA repair and cancer. However, the functional detail and mechanistic regulation of macroH2A in the DDR pathway is poorly understood. In our preliminary study, we identified ZMYM3 (Zinc finger myeloproliferative and mental retardation, type-3) as a novel macroH2A interacting protein, which may participate in regulating the DDR. In this proposal, we will use genetic and proteomic approaches to address the fundamental question of how macroH2A-ZMYM3 are involved in promoting DNA repair. We will utilize genetic engineered cell lines to study the signaling regulations of macroH2A-ZMYM3 axis in the DDR pathway. We will perform proteomic analysis to define the macroH2A-ZMYM3 network and ZMYM3 functional complex that is involved in the DDR. This project will provide mechanistic insights into the DDR regulation in the context of chromatin and macroH2A-mediated pathways. These studies will also overcome previous limitations associated with studying histone variants in human cells. Our preliminary data shows that macroH2A is a substrate of DDR ubiquitin E3 ligase (RNF168) and is required for cell survival upon DNA damage. In the proposed study, we will first investigate the role of macroH2A in various DNA damage responses using macroH2A or/and H2AX deficient cells. Our hypothesis is that macroH2A mediates the DDR pathway by facilitating repair protein recruitment to the double strand break site, distinct from H2AX. We will also characterize the roles the newly identified macroH2A binding protein, ZMYM3, in the DDR pathway. In addition, we propose to study the ZMYM3 cellular functions and the mechanism of macroH2A-ZMYM3 protein network in regulating homologous recombination repair. Finally, building upon our preliminary studies from our genetic system, we will elucidate the role of macroH2A-ZMYM3 in tumorigenesis. These studies will provide mechanistic insight of how macroH2A orchestrates the DDR pathway to protect the genome integrity. Many cancer treatment strategies rely on DNA damaging agents, including chemotherapeutic agents and radiotherapy. Identifying new DNA repair proteins will provide potential cancer biomarkers and novel targets for drug discovery. Thus, this proposal exploits a combination of genetic, cellular and biochemical approaches in human cells to dissect the unsolved puzzle of genome integrity maintenance, potentially advancing cancer therapies including.